Treatment-naïve HPV+ head and neck cancers display a T-cell-inflamed phenotype distinct from their HPV- counterparts that has implications for immunotherapy

Abstract

Cancers progress when the immune system fails to identify and eliminate malignant cells. Recognition of this, combined with advances in tumor immunology, has allowed development of therapies that induce effective anti-tumor immune responses. For incompletely-understood reasons, effective responses to immunotherapy occur in some patients and not others. Head and neck squamous cell carcinomas (HNSCC) are a common cancer type that can be divided into two subsets based on human papillomavirus (HPV) status. HPV status is a strong predictor of positive clinical outcome. Expression of exogenous viral antigens by HPV+, but not HPV-, HNSCC allows direct comparison of the immune status (immune cell presence and characteristics) between these two otherwise anatomically-similar tumors. Using TCGA data, we compared the immune landscape between HPV+ and HPV- treatment-naïve HNSCC. As compared to HPV- samples, HPV+ HNSCC exhibited a strong Th1 response characterized by increased infiltration with multiple types of immune cells and expression of their effector molecules. HPV+ HNSCC also expressed higher levels of CD39 and multiple T-cell exhaustion markers including LAG3, PD1, TIGIT, and TIM3 compared to HPV- HNSCC. Importantly, patients with higher expression of these exhaustion markers-indicative of a T-cell-inflamed tumor-correlated with markedly improved survival in HPV+, but not HPV-, HNSCC. Thus, profound differences exist between the immune landscape of HPV+ and HPV- HNSCC. These results suggest that immune checkpoint inhibitor therapy is a promising treatment strategy for HPV+ HNSCC, and that expression of immune checkpoint molecules could serve as a predictive biomarker of patient outcome in HPV+ HNSCC.

Keywords: head and neck cancer; human papillomavirus; immune checkpoint markers; survival; tumour infiltrating lymphocytes.

Figure 1.

The relative population of 22 leukocyte types present in the tumor microenvironment was determined by the CIBERSORT algorithm and compared between HPV+, HPV-, and normal control samples. Four leukocyte types (neutrophils, eosinophils, naïve CD4 ⁺ T-cells, and gamma delta T-cells) are not shown, as they were undetectable in this cohort. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001, ns (not significant).

Figure 2.

Expression of marker genes indicating that CD4⁺ helper T-cell subsets present in head & neck carcinomas stratified by human papillomavirus (HPV) status. Normalized RNA-seq data for TBX21 (Th1), GATA3 and STAT6 (Th2), and RORA and RORC (Th17) was extracted from The Cancer Genome Atlas (TCGA) database for the head & neck cancer (HNSC) cohort for HPV+, HPV−, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001, ns (not significant).

Figure 3.

Detection of tumor infiltrating T-cells and their activation status in head & neck carcinomas stratified by HPV status. Normalized RNA-seq data for genes indicative of tumor infiltrating CD8⁺ (CD8A and CD8B) or CD4⁺ (CD4) T-cells and their activation status (CD137, INFG, and TNF) was extracted from the TCGA database for the HNSC cohort for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns (not significant).

Figure 4.

Transcript levels of tumor-derived interferon-responsive immunomodulatory genes in head & neck carcinomas stratified by HPV status. Normalized RNA-seq data for genes associated with tumor cell mediated immunomodulation including IDO1, its negative regulator BIN1, and PDL1 was extracted from the TCGA database for the HNSC cohort for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns (not significant).

Figure 5.

Detection of dendritic cells and their effector cytokines in head & neck carcinomas, stratified by HPV status. Normalized RNA-seq data for genes indicative of tumor infiltrating dendritic cells (CD103 and CD11C) and genes encoding cytokines indicative of dendritic cell activation status (IL12A, STAT4, IL23A, and EBI3) was extracted from the TCGA database for the HNSC cohort for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns (not significant).

Figure 6.

Transcript levels of T-cell exhaustion marker genes and CD39 in head & neck carcinomas, stratified by HPV status. Normalized RNA-seq data for genes associated with T-cell exhaustion (LAG3, PD1, TIGIT, TIM3, and CD39) was extracted from the TCGA database for the HNSC cohort for HPV+, HPV-, and normal control tissues. Numbers in brackets refer to the number of samples included in each analysis. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns (not significant).

Figure 7.

High LAG3, PD1, TIGIT, or TIM3 gene transcript levels are strongly associated with improved survival in treatment naïve patients with HPV+ but not HPV- head & neck carcinomas. Overall survival of patients within the HPV-positive cohort dichotomized by median expression of LAG3, PD1, TIGIT, and TIM3. Comparison between groups was made by the 2-sided log-rank test. Red = low expression of the indicated gene in HPV+ samples, Black = high expression of the indicated gene in HPV+ samples, Purple = low expression of the indicated gene in HPV- samples, Green = high expression of the indicated gene in HPV- samples.

Figure 8.

Concordant levels of multiple markers of T-cell exhaustion is strongly associated with survival in patients with HPV+ head & neck carcinomas. Overall survival of patients within the HPV-positive cohort dichotomized by median transcript levels of the indicated pairs of T-cell exhaustion markers. Comparison between groups was made by the 2-sided log-rank test. Red = low expression of the indicated gene in HPV+ samples, Black = high expression of the indicated gene in HPV+ samples, Purple = low expression of the indicated gene in HPV- samples, Green = high expression of the indicated gene in HPV- samples.

Figure 9.

High CD39 gene transcript level is strongly associated with improved survival in treatment naïve patients with HPV+head & neck carcinomas. Overall survival of patients within the HPV+ and HPV- cohorts were dichotomized by median expression of CD39. Concordant levels of CD39 and multiple markers of T-cell exhaustion was strongly associated with survival in patients with HPV+ head & neck carcinomas. Overall survival of patients within the HPV-positive cohort dichotomized by median transcript levels of the indicated pairs of T-cell exhaustion markers. Comparison between groups was made by the 2-sided log-rank test. Red = low expression of the indicated gene in HPV+ samples, Black = high expression of the indicated gene in HPV+ samples, Purple = low expression of the indicated gene in HPV- samples, Green = high expression of the indicated gene in HPV- samples.